Appendix J
J. Effect of cactus RNAi (250ng) on knockdown efficiency and AMP gene expression to Candida
time 0.01 0.01 1.08 0.29
RNAi*time -0.03 0.02 -1.42 0.16
Constitutive (time 0)
cactus Intercept -3.86 0.45 -8.62 5.69E-07
RNAi
treatment -4.70 0.63 -7.42 3.27E-06
defensin-2 Intercept -0.37 0.54 -0.68 0.51
RNAi
treatment -4.90 0.76 -6.44 1.54E-05
defensin-3 Intercept -3.87 0.44 -8.84 4.22E-07
RNAi
treatment -3.03 0.62 -4.89 2.37E-04
cecropin-2 Intercept -3.86 0.45 -8.62 5.69E-07
RNAi
treatment -4.70 0.63 -7.42 3.27E-06 Resolution (time 48)
cactus Intercept -3.52 0.57 -6.15 2.51E-05
RNAi
treatment -3.29 0.81 -4.06 1.16E-03
defensin-2 Intercept -0.96 0.31 -3.10 7.80E-03
RNAi
treatment -1.69 0.44 -3.86 1.73E-03
defensin-3 Intercept -4.15 0.52 -8.03 1.30E-06
RNAi
treatment -3.67 0.73 -5.02 1.87E-04
cecropin-2 Intercept -3.52 0.57 -6.15 2.51E-05
RNAi
treatment -3.29 0.81 -4.06 1.16E-03
Appendix K
K. Effect of cactus RNAi dose on knockdown efficiency and AMP gene expression to Bacillus thuringiensis infection in flour beetles compared to MalE control. Linear models (expression ~ dose*time) conducted with
"lm" function in R. Time denotes the hours passed post microbial challenge while dose represents the effect of cactus dsRNA per ng. Expression values are on a log scale. P values less than the significance threshold after correcting for multiple testing (Bonferroni method; α = 0.0167) are in bold.
Gene Factor Estimate Std. Error t value Pval
cactus Intercept -2.64 0.06 -46.06 < 2e-16
dose 0.00 0.00 -3.61 3.89E-04
time -0.01 0.00 -4.13 5.50E-05
dose*time 0.00 0.00 0.27 0.79
Induction (times 0-6)
defensin-2 Intercept -2.70 0.36 -7.44 5.20E-11
dose 0.01 0.00 3.60 5.23E-04
time 0.48 0.10 4.78 6.51E-06
dose*time 0.00 0.00 -1.85 0.07
defensin-3 Intercept -6.24 0.24 -25.82 < 2e-16
dose 0.01 0.00 3.90 1.85E-04
time 0.22 0.07 3.30 1.38E-03
dose*time 0.00 0.00 -1.27 0.21
cecropin-2 Intercept -8.13 0.34 -23.69 < 2e-16
dose 0.01 0.00 5.26 9.37E-07
time 0.10 0.09 1.04 0.30
dose*time 0.00 0.00 -0.92 0.36
Decay (times 12-48)
defensin-2 Intercept 0.73 0.35 2.12 0.04
dose 0.00 0.00 0.66 0.51
time -0.07 0.01 -6.71 1.71E-09
dose*time 0.00 0.00 2.16 0.03
defensin-3 Intercept -4.96 0.37 -13.55 <2e-16
dose 0.01 0.00 1.84 0.07
time -0.06 0.01 -4.86 4.93E-06
dose*time 0.00 0.00 1.33 0.19
cecropin-2 Intercept -6.89 0.51 -13.53 <2e-16
dose 0.01 0.00 1.27 0.21
time -0.01 0.02 -0.63 0.53
dose*time 0.00 0.00 1.48 0.14
Constitutive (time 0)
cactus Intercept -2.82 0.10 -28.85 <2e-16
dose 0.00 0.00 -2.22 3.41E-02
defensin-2 Intercept -3.87 0.43 -9.02 4.76E-10
dose 0.01 0.00 4.39 1.30E-04
defensin-3 Intercept -6.67 0.29 -22.98 <2e-16
dose 0.01 0.00 3.84 5.99E-04
cecropin-2 Intercept -8.19 0.34 -24.41 <2e-16
dose 0.02 0.00 5.94 1.67E-06
Resolution (time 48)
cactus Intercept -3.22 0.09 -34.65 <2e-16
dose 0.00 0.00 -1.14 0.26
defensin-2 Intercept -2.52 0.31 -8.23 3.47E-09
dose 0.01 0.00 4.08 3.10E-04
defensin-3 Intercept -7.41 0.33 -22.64 <2e-16
dose 0.01 0.00 4.19 2.25E-04
cecropin-2 Intercept -7.33 0.53 -13.83 1.49E-14
dose 0.01 0.00 3.16 3.60E-03
Appendix L
L. Effect of cactus RNAi (250ng) on total circulating hemocytes. Full factorial Wilcoxon rank sum test on total circulating hemocyte counts. Conducted with "wilcox.test" function from the stats
package in R. Post-hoc FDR correction for multiple comparisons ushamng the p.adjust function in R.
shamgnificant P values are in bold (α = 0.05). Time denotes the hours passed post-challenge, with 0 hours indicating the period before any challenge.
Treatment 1
Time 1
Infection 1
Treatment 2
Time 2
Infection
2 Pval Pval.adjusted
Cactus 6 Bt Cactus 12 Bt 1.19E-05 1.56E-04
Cactus 6 Bt MalE 0 Naive 8.23E-05 2.21E-04
Cactus 6 Bt Cactus 0 Naive 8.23E-05 2.21E-04
Cactus 6 Bt Cactus 24 Bt 2.26E-04 3.72E-04
Cactus 6 Bt MalE 12 Bt 1.45E-03 2.11E-03
Cactus 6 Bt MalE 170 Bt 2.99E-03 4.16E-03
Cactus 6 Bt MalE 24 Bt 0.14 0.16
Cactus 6 Bt Cactus 170 Bt 0.50 0.53
Cactus 6 sham Cactus 12 Bt 2.65E-05 1.56E-04
Cactus 6 sham Cactus 24 Bt 2.65E-05 1.56E-04
Cactus 6 sham Cactus 170 Bt 2.65E-05 1.56E-04
Cactus 6 sham MalE 12 Bt 4.57E-05 1.84E-04
Cactus 6 sham Cactus 6 Bt 8.23E-05 2.21E-04
Cactus 6 sham MalE 24 Bt 8.23E-05 2.21E-04
Cactus 6 sham MalE 12 sham 1.55E-04 2.58E-04
Cactus 6 sham Cactus 12 sham 1.55E-04 2.58E-04
Cactus 6 sham Cactus 24 sham 1.55E-04 2.58E-04
Cactus 6 sham MalE 0 Naive 1.55E-04 2.58E-04
Cactus 6 sham Cactus 0 Naive 1.55E-04 2.58E-04
Cactus 6 sham MalE 24 sham 3.11E-04 5.06E-04
Cactus 6 sham MalE 170 Bt 4.39E-03 5.99E-03
Cactus 6 sham MalE 6 Bt 2.06E-02 2.59E-02
Cactus 6 sham MalE 170 sham 0.19 0.22
Cactus 6 sham Cactus 170 sham 0.19 0.22
Cactus 12 Bt MalE 170 Bt 5.67E-06 1.56E-04
Cactus 12 Bt Cactus 170 Bt 5.67E-06 1.56E-04
Cactus 12 Bt MalE 0 Naive 2.65E-05 1.56E-04
Cactus 12 Bt Cactus 0 Naive 2.65E-05 1.56E-04
Cactus 12 Bt MalE 24 Bt 1.43E-04 2.58E-04
Cactus 12 Bt Cactus 24 Bt 0.17 0.20
Cactus 12 sham MalE 170 Bt 4.57E-05 1.84E-04
Cactus 12 sham MalE 6 Bt 8.23E-05 2.21E-04
Cactus 12 sham MalE 170 sham 1.55E-04 2.58E-04
Cactus 12 sham Cactus 170 sham 1.55E-04 2.58E-04
Cactus 12 sham MalE 0 Naive 1.55E-04 2.58E-04
Cactus 12 sham Cactus 0 Naive 1.55E-04 2.58E-04
Cactus 12 sham Cactus 6 Bt 5.76E-04 8.99E-04
Cactus 12 sham Cactus 170 Bt 3.84E-03 5.29E-03
Cactus 12 sham Cactus 12 Bt 4.97E-03 6.68E-03
Cactus 12 sham MalE 24 sham 1.04E-02 1.34E-02
Cactus 12 sham Cactus 24 sham 0.06 0.08
Cactus 12 sham Cactus 24 Bt 0.11 0.13
Cactus 12 sham MalE 24 Bt 0.17 0.19
Cactus 12 sham MalE 12 Bt 0.90 0.90
Cactus 24 Bt MalE 170 Bt 1.13E-05 1.56E-04
Cactus 24 Bt MalE 0 Naive 2.65E-05 1.56E-04
Cactus 24 Bt Cactus 0 Naive 2.65E-05 1.56E-04
Cactus 24 Bt Cactus 170 Bt 1.28E-04 2.58E-04
Cactus 24 sham MalE 170 sham 1.55E-04 2.58E-04
Cactus 24 sham Cactus 170 sham 1.55E-04 2.58E-04
Cactus 24 sham Cactus 12 Bt 1.06E-04 2.58E-04
Cactus 24 sham MalE 0 Naive 1.55E-04 2.58E-04
Cactus 24 sham Cactus 0 Naive 1.55E-04 2.58E-04
Cactus 24 sham MalE 6 Bt 9.87E-04 1.50E-03
Cactus 24 sham MalE 170 Bt 1.37E-03 2.02E-03
Cactus 24 sham Cactus 24 Bt 2.54E-03 3.63E-03
Cactus 24 sham MalE 12 Bt 0.07 0.08
Cactus 24 sham Cactus 6 Bt 0.17 0.19
Cactus 24 sham Cactus 170 Bt 0.44 0.47
Cactus 24 sham MalE 24 Bt 0.74 0.76
Cactus 170 Bt MalE 0 Naive 2.65E-05 1.56E-04
Cactus 170 Bt Cactus 0 Naive 2.65E-05 1.56E-04
Cactus 170 sham Cactus 12 Bt 2.65E-05 1.56E-04
Cactus 170 sham Cactus 24 Bt 2.65E-05 1.56E-04
Cactus 170 sham Cactus 170 Bt 2.65E-05 1.56E-04
Cactus 170 sham MalE 12 Bt 4.57E-05 1.84E-04
Cactus 170 sham Cactus 6 Bt 8.23E-05 2.21E-04
Cactus 170 sham MalE 24 Bt 8.23E-05 2.21E-04
Cactus 170 sham MalE 170 Bt 9.14E-05 2.41E-04
Cactus 170 sham MalE 0 Naive 1.55E-04 2.58E-04
Cactus 170 sham Cactus 0 Naive 1.55E-04 2.58E-04
Cactus 170 sham MalE 6 Bt 9.87E-04 1.50E-03
MalE 0 Naive Cactus 0 Naive 0.43 0.47
MalE 6 Bt MalE 12 Bt 2.17E-05 1.56E-04
MalE 6 Bt Cactus 12 Bt 1.19E-05 1.56E-04
MalE 6 Bt Cactus 24 Bt 1.19E-05 1.56E-04
MalE 6 Bt MalE 0 Naive 8.23E-05 2.21E-04
MalE 6 Bt Cactus 0 Naive 8.23E-05 2.21E-04
MalE 6 Bt Cactus 170 Bt 3.57E-04 5.63E-04
MalE 6 Bt Cactus 6 Bt 2.76E-03 3.88E-03
MalE 6 Bt MalE 170 Bt 0.28 0.31
MalE 6 sham Cactus 12 Bt 2.65E-05 1.56E-04
MalE 6 sham Cactus 24 Bt 2.65E-05 1.56E-04
MalE 6 sham Cactus 170 Bt 2.65E-05 1.56E-04
MalE 6 sham MalE 12 Bt 4.57E-05 1.84E-04
MalE 6 sham MalE 170 Bt 4.57E-05 1.84E-04
MalE 6 sham MalE 6 Bt 8.23E-05 2.21E-04
MalE 6 sham Cactus 6 Bt 8.23E-05 2.21E-04
MalE 6 sham MalE 24 Bt 8.23E-05 2.21E-04
MalE 6 sham MalE 12 sham 1.55E-04 2.58E-04
MalE 6 sham Cactus 12 sham 1.55E-04 2.58E-04
MalE 6 sham MalE 24 sham 1.55E-04 2.58E-04
MalE 6 sham Cactus 24 sham 1.55E-04 2.58E-04
MalE 6 sham MalE 0 Naive 1.55E-04 2.58E-04
MalE 6 sham Cactus 0 Naive 1.55E-04 2.58E-04
MalE 6 sham Cactus 170 sham 0.34 0.38
MalE 6 sham MalE 170 sham 0.46 0.49
MalE 6 sham Cactus 6 sham 0.88 0.89
MalE 12 Bt MalE 170 Bt 1.08E-05 1.56E-04
MalE 12 Bt MalE 0 Naive 4.57E-05 1.84E-04
MalE 12 Bt Cactus 0 Naive 4.57E-05 1.84E-04
MalE 12 Bt Cactus 12 Bt 2.76E-03 3.88E-03
MalE 12 Bt Cactus 170 Bt 4.79E-03 6.48E-03
MalE 12 Bt Cactus 24 Bt 0.13 0.16
MalE 12 Bt MalE 24 Bt 0.16 0.19
MalE 12 sham MalE 170 Bt 4.57E-05 1.84E-04
MalE 12 sham MalE 6 Bt 8.23E-05 2.21E-04
MalE 12 sham MalE 170 sham 1.55E-04 2.58E-04
MalE 12 sham Cactus 170 sham 1.55E-04 2.58E-04
MalE 12 sham MalE 0 Naive 1.55E-04 2.58E-04
MalE 12 sham Cactus 0 Naive 1.55E-04 2.58E-04
MalE 12 sham Cactus 12 Bt 3.18E-04 5.06E-04
MalE 12 sham Cactus 6 Bt 2.47E-03 3.56E-03
MalE 12 sham Cactus 170 Bt 1.32E-02 1.68E-02
MalE 12 sham Cactus 24 Bt 2.03E-02 2.57E-02
MalE 12 sham MalE 24 sham 3.79E-02 4.72E-02
MalE 12 sham Cactus 24 sham 0.19 0.22
MalE 12 sham Cactus 12 sham 0.44 0.47
MalE 12 sham MalE 24 Bt 0.48 0.51
MalE 12 sham MalE 12 Bt 0.53 0.56
MalE 24 Bt MalE 0 Naive 8.23E-05 2.21E-04
MalE 24 Bt Cactus 0 Naive 8.23E-05 2.21E-04
MalE 24 Bt MalE 170 Bt 6.50E-04 1.00E-03
MalE 24 Bt Cactus 24 Bt 5.66E-03 7.53E-03
MalE 24 Bt Cactus 170 Bt 0.29 0.33
MalE 24 sham Cactus 12 Bt 5.29E-05 2.08E-04
MalE 24 sham MalE 170 sham 1.55E-04 2.58E-04
MalE 24 sham Cactus 170 sham 1.55E-04 2.58E-04
MalE 24 sham MalE 0 Naive 1.55E-04 2.58E-04
MalE 24 sham Cactus 0 Naive 1.55E-04 2.58E-04
MalE 24 sham Cactus 24 Bt 3.18E-04 5.06E-04
MalE 24 sham MalE 6 Bt 7.90E-03 1.04E-02
MalE 24 sham MalE 12 Bt 8.55E-03 1.11E-02
MalE 24 sham MalE 170 Bt 8.55E-03 1.11E-02
MalE 24 sham MalE 24 Bt 0.17 0.19
MalE 24 sham Cactus 24 sham 0.34 0.38
MalE 24 sham Cactus 170 Bt 0.72 0.74
MalE 24 sham Cactus 6 Bt 0.81 0.83
MalE 170 Bt MalE 0 Naive 4.57E-05 1.84E-04
MalE 170 Bt Cactus 0 Naive 4.57E-05 1.84E-04
MalE 170 Bt Cactus 170 Bt 1.19E-03 1.79E-03
MalE 170 sham Cactus 12 Bt 2.65E-05 1.56E-04
MalE 170 sham Cactus 24 Bt 2.65E-05 1.56E-04
MalE 170 sham Cactus 170 Bt 2.65E-05 1.56E-04
MalE 170 sham MalE 12 Bt 4.57E-05 1.84E-04
MalE 170 sham MalE 170 Bt 4.57E-05 1.84E-04
MalE 170 sham MalE 6 Bt 8.23E-05 2.21E-04
MalE 170 sham Cactus 6 Bt 8.23E-05 2.21E-04
MalE 170 sham MalE 24 Bt 8.23E-05 2.21E-04
MalE 170 sham MalE 0 Naive 1.55E-04 2.58E-04
MalE 170 sham Cactus 0 Naive 1.55E-04 2.58E-04
MalE 170 sham Cactus 170 sham 1.00 1.00
Appendix M